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Lymph Nodes: How to Obtain Excellent Specimens

Kimberly J. Caruso, DVM

Clinical Pathology

November 2003
Peer Reviewed

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Lymph nodes are among the most commonly sampled organs for cytologic examination of lymphadenopathy. Diagnostic classifications of enlarged lymph nodes are benign reactive hyperplasia, lymphoma, lymphadenitis, and metastatic neoplasia. In addition to distinguishing between benign and malignant conditions, cytologic evaluation also enables identification of specific etiological agents, such as Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, Neorickettsia helminthoeca, Mycobacterium species, and leishmaniasis. Such evaluation is cost-effective and simple, and sample collection is reasonably well-tolerated by the patient.

The ease with which lymphoid cells exfoliate, their relative fragility, and the proximity of lymphoid tissue to other tissues can affect sample quality. Inadequate or misleading specimens include those of low cellularity, those consisting of ruptured lymphoid cells, smears that are too thick, and smears that consist of nonlymphoid tissue such as salivary gland tissue or perinodal fat. The following questions and answers will help ensure acquisition of a quality specimen.

Q • Which nodes should I aspirate and how many smears should I make? A • Samples should be obtained from all enlarged nodes. Slides should be accurately labeled with which lymph node has been sampled, since different diagnoses may be obtained. Because the submandibular lymph nodes are often reactive secondary to periodontal disease, aspiration of these nodes is less desirable because the hyperplasia may confuse the cytologic picture. Unless these lymph nodes are the only ones enlarged, aspiration of other nodes is preferred. Try to submit at least two smears from each site.

Q • Which technique do you prefer for collecting cytologic specimens? A • I generally prefer the nonaspiration technique, which minimizes peripheral blood contamination and cell rupture secondary to the suction trauma induced by aspiration. Rupture is common in lymph node cell collection because of their fragility. The nonaspiration technique typically yields well-preserved samples of high cellularity. Using a 22-gauge needle, isolate the tissue of interest with one hand and insert the needle into the tissue. Redirect the needle several times without exiting the skin. This facilitates sampling of several areas of the tissue, maximizing the chance of obtaining a representative sample. Attach an air-filled 6-ml syringe to the hub of the needle. Aim the needle at the middle of a glass microscope slide with the needle almost touching the slide. Expel the air. Immediately lay another slide over the first slide in the same direction, approximately three quarters of the way. Gently pull the slides apart in a horizontal fashion. You will have two smears. Allow them to air-dry. Do not heat-fix-this step is unnecessary for cytologic specimens and often introduces artifacts. Do not ship these slides along with jars of formalin-fixed tissues-formalin fumes interfere with the ability of the cells to absorb the stain.  

Q • What other information should I include with the samples? A • Don't forget to include the signalment of the animal as well as clinical history, current therapy, diseases suspected, the number of lymph nodes involved, and which lymph node(s) was sampled. Each slide should also be labeled with the name of the patient as well as the site of the lymph node if several nodes have been sampled.  

Q • Once I have obtained my smears, how can I tell if they are representative and diagnostic? A • Stain one of them using a rapid modified Wright's stain (i.e., Diff-Quik, Dip Stat). Allow to air-dry. Smears from lymph nodes grossly appear blue. Using 10X magnification, evaluate the cellularity by asking, "Are cells present?"

For adequate cellularity, moderate to high numbers of lymphoid cells should be present. Then ask, "Are these cells lymphoid cells?" Lymphoid cells comprise the main cell population in lymph node aspirates and appear round, with a round, purple nucleus and a small amount of encircling cytoplasm. These cells may vary in size and type from small lymphocytes (1 to 1.5 red blood cells in diameter) (Figure 1A) to more immature prolymphocytes (Figure 1B) and lymphoblasts (3 to 5 red blood cells in diameter) (Figure 1C). Once you have confirmed that the sample has satisfactory cellularity, ask yourself, "Are most of the cells intact?" Intact cells have visible cytoplasm with defined cytoplasmic margins. Do not mistake bare nuclei for an intact cell (Figures 2 and 3). Remember, adequate interpretation of a cytologic smear from a lymph node is dependent on an intact cell population.

Q • What if I am unable to recognize the tissue I have aspirated? A • If you observe cells not compatible with lymph node tissue, adipose tissue, or salivary gland tissue, submit the smears to a clinical pathologist for evaluation and interpretation. The enlargement may be a mass in proximity to a lymph node or metastatic disease involving the entire lymph node (Figure 4).  

Q • What normal tissues have the potential to be accidentally aspirated along with the lymph node cells? How can I differentiate them from lymphoid tissue? A • Perinodal fat (especially in obese animals) and salivary gland tissue (if aspirating the submandibular lymph nodes) are the normal tissues that can be aspirated when trying to obtain a lymph node sample. Perinodal fat may appear as glistening, clear material on a smear before staining. When stained, its appearance may range from ruptured fat droplets to clusters of adipocytes, which appear as large, round, clear cells with a small, flattened purple nucleus pushed off to the side of the cell (Figure 5). Salivary gland tissue (Figure 6) appears as clusters of epithelial cells with abundant finely vacuolated cytoplasm. Saliva may be present when the material on the slide seems sticky because of saliva's viscous nature. Saliva may also be present when the aspirated red blood cells line up in rows due to the viscosity from the saliva. In the case of obtaining perinodal fat or a salivary gland or saliva, digitally relocate the lymph node and repeat the procedure using a new needle and slides.


Do not ship these slides along with jars of formalin-fixed tissues-formalin fumes interfere with the ability of the cells to absorb the stain.

1A. Low-power magnification of sample from a reactive lymph node. Note the high numbers of lymphoid cells present individually, which indicates adequate specimen sampling.

1B. The smear of this sample is too thick and inadequately spread, making the cells unidentifiable.

1C. Figure 1A at greater magnification. Note that most of the lymphoid cells are intact, and the nuclear chromatin stains darkly on the small mature lymphocytes (arrow) with a minimal rim of basophilic cytoplasm. Low numbers of lymphoblasts and an occasional plasma cell are also noted in this smear, demonstrating mildly reactive lymphoid hyperplasia. The lymphoblast (*) has a larger nucleus with less condensed chromatin that stains a lighter purple, is also surrounded by a thin rim of basophilic cytoplasm, and often demonstrates a single nucleolus. The plasma cell (x) has an eccentrically placed dark purple nucleus, a moderate amount of encircling deeper basophilic cytoplasm, and often a paranuclear clear zone.

Aspirate from a dog's popliteal lymph node. The cells are intact but not recognizable as normal lymphoid tissue. This is a case of metastatic melanoma. Note the numerous greenish granules (melanin) present in the cytoplasm within the melanocytes and melanophages.

Contact a diagnostic laboratory for additional advice if you routinely have problems obtaining diagnostic specimens. Laboratory personnel are usually very willing to cooperate to ensure that the specimen yields as much information as possible.

Contact a diagnostic laboratory for additional advice if you routinely have problems obtaining diagnostic specimens. Laboratory personnel are usually very willing to cooperate to ensure that the specimen yields as much information as possible.

Normal salivary gland at both low-power (A) and high-power (B) magnification. Note the large and smaller clusters of the salivary gland epithelial cells; the highly vacuolated nature of the cytoplasm; and the dense, purple nuclei.


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